Department of Psychiatry and Behavioral Sciences, Center for Neuroscience, California National Primate Research Center and The MIND Institute, University of California-Davis, 2825 50th Street, Sacramento, CA 95817, USA.

ABSTRACT Autism together with Asperger syndrome and pervasive developmental disorder not otherwise specified form a spectrum of conditions (autism spectrum disorders or ASD) that is characterized by disturbances in social behavior, impaired communication and the presence of stereotyped behaviors or circumscribed interests. Recent estimates indicate a prevalence of ASD of 1 per 150 (Kuehn, 2007). The cause(s) of most cases of ASD are unknown but there is an emerging consensus that ASD have multiple etiologies. One proposed cause of ASD is exposure of the fetal brain to maternal autoantibodies during pregnancy [Dalton, P., Deacon, R., Blamire, A., Pike, M., McKinlay, I., Stein, J., Styles, P., Vincent, A., 2003. Maternal neuronal antibodies associated with autism and a language disorder. Ann. Neurol. 53, 533-537]. To provide evidence for this hypothesis, four rhesus monkeys were exposed prenatally to human IgG collected from mothers of multiple children diagnosed with ASD. Four control rhesus monkeys were exposed to human IgG collected from mothers of multiple typically developing children. Five additional monkeys were untreated controls. Monkeys were observed in a variety of behavioral paradigms involving unique social situations. Behaviors were scored by trained observers and overall activity was monitored with actimeters. Rhesus monkeys gestationally exposed to IgG class antibodies from mothers of children with ASD consistently demonstrated increased whole-body stereotypies across multiple testing paradigms. These monkeys were also hyperactive compared to controls. Treatment with IgG purified from mothers of typically developing children did not induce stereotypical or hyperactive behaviors. These findings support the potential for an autoimmune etiology in a subgroup of patients with neurodevelopmental disorders. This research raises the prospect of prenatal evaluation for neurodevelopmental risk factors and the potential for preventative therapeutics.

[Show abstract][Hide abstract]ABSTRACT:
Recent studies of Autism Spectrum Disorders (ASD) highlight hyperactivity of the immune system, irregular neuronal growth and increased size and number of microglia. Though the small sample size in many of these studies limits extrapolation to all individuals with ASD, there is mounting evidence of both immune and nervous system related pathogenesis in at least a subset of patients with ASD. Given the disturbing rise in incidence rates for ASD, and the fact that no pharmacological therapy for ASD has been approved by the Food and Drug Administration (FDA), there is an urgent need for new therapeutic options. Research in the therapeutic effects of mesenchymal stem cells (MSC) for other immunological and neurological conditions has shown promising results in preclinical and even clinical studies. MSC have demonstrated the ability to suppress the immune system and to promote neurogenesis with a promising safety profile. The working hypothesis of this paper is that the potentially synergistic ability of MSC to modulate a hyperactive immune system and its ability to promote neurogenesis make it an attractive potential therapeutic option specifically for ASD. Theoretical mechanisms of action will be suggested, but further research is necessary to support these hypothetical pathways. The choice of tissue source, type of cell, and most appropriate ages for therapeutic intervention remain open questions for further consideration. Concern over poor regulatory control of stem cell studies or treatment, and the unique ethical challenges that each child with ASD presents, demands that future research be conducted with particular caution before widespread use of the proposed therapeutic intervention is implemented.

[Show abstract][Hide abstract]ABSTRACT:
Increasing epidemiological and experimental evidence implicates gestational infections as one important factor involved in the pathogenesis of several neuropsychiatric disorders. Corresponding preclinical model systems based upon maternal immune activation (MIA) by treatment of the pregnant female have been developed. These MIA animal model systems have been successfully used in basic and translational research approaches, contributing to the investigation of the underlying pathophysiological mechanisms at the molecular, cellular and behavioural levels. The present article focusses on the application of a specific MIA rodent paradigm, based upon treatment of the gestating dam with the viral mimic polyinosinic-poly cytidilic acid (Poly(I:C)), a synthetic analog of double-stranded RNA (dsRNA) which activates the Toll-like receptor 3 (TLR3) pathway. Important advantages and constraints of this animal model will be discussed, specifically in light of gestational infection as one vulnerability factor contributing to the complex aetiology of mood and psychotic disorders, which are likely the result of intricate multi-level gene x environment interactions. Improving our currently incomplete understanding of the molecular pathomechanistic principles underlying these disorders is a prerequisite for the development of alternative therapeutic approaches which are critically needed in light of the important drawbacks and limitations of currently available pharmacological treatment options regarding efficacy and side effects. The particular relevance of the Poly(I:C) MIA model for the discovery of novel drug targets for symptomatic and preventive therapeutic strategies in mood and psychotic disorders is highlighted in this review article.

[Show abstract][Hide abstract]ABSTRACT:
The nervous and immune systems have evolved in parallel from the early bilaterians, in which innate immunity and a central nervous system (CNS) coexisted for the first time, to jawed vertebrates and the appearance of adaptive immunity. The CNS feeds from, and integrates efferent signals in response to, somatic and autonomic sensory information. The CNS receives input also from the periphery about inflammation and infection. Cytokines, chemokines, and damage-associated soluble mediators of systemic inflammation can also gain access to the CNS via blood flow. In response to systemic inflammation, those soluble mediators can access directly through the circumventricular organs, as well as open the blood-brain barrier. The resulting translocation of inflammatory mediators can interfere with neuronal and glial well-being, leading to a break of balance in brain homeostasis. This in turn results in cognitive and behavioral manifestations commonly present during acute infections - including anorexia, malaise, depression, and decreased physical activity - collectively known as the sickness behavior (SB). While SB manifestations are transient and self-limited, under states of persistent systemic inflammatory response the cognitive and behavioral changes can become permanent. For example, cognitive decline is almost universal in sepsis survivors, and a common finding in patients with systemic lupus erythematosus. Here, we review recent genetic evidence suggesting an association between neurodegenerative disorders and persistent immune activation; clinical and experimental evidence indicating previously unidentified immune-mediated pathways of neurodegeneration; and novel immunomodulatory targets and their potential relevance for neurodegenerative disorders.

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Stereotypies and hyperactivity in rhesus monkeys exposed to IgGfrom mothers of children with autismqLoren A. Martina,1, Paul Ashwoodb,c, Daniel Braunschweigd, Maricel Cabanlitd,Judy Van de Waterc,d, David G. Amarala,c,*aDepartment of Psychiatry and Behavioral Sciences, Center for Neuroscience, California National Primate Research Center and The M.I.N.D.Institute, University of California-Davis, 2825 50th Street, Sacramento, CA 95817, USAbDepartment of Medical Microbiology and Immunology and the M.I.N.D. Institute, University of California, Davis, CA, USAcNIEHS Center for Children’s Environmental Health, University of California, Davis, CA, USAdDivision of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, CA, USAReceived 23 September 2007; received in revised form 16 December 2007; accepted 22 December 2007Available online 8 February 2008AbstractAutism together with Asperger syndrome and pervasive developmental disorder not otherwise specified form a spectrum of con-ditions (autism spectrum disorders or ASD) that is characterized by disturbances in social behavior, impaired communication andthe presence of stereotyped behaviors or circumscribed interests. Recent estimates indicate a prevalence of ASD of 1 per 150(Kuehn, 2007). The cause(s) of most cases of ASD are unknown but there is an emerging consensus that ASD have multiple eti-ologies. One proposed cause of ASD is exposure of the fetal brain to maternal autoantibodies during pregnancy [Dalton, P., Dea-con, R., Blamire, A., Pike, M., McKinlay, I., Stein, J., Styles, P., Vincent, A., 2003. Maternal neuronal antibodies associated withautism and a language disorder. Ann. Neurol. 53, 533–537]. To provide evidence for this hypothesis, four rhesus monkeys wereexposed prenatally to human IgG collected from mothers of multiple children diagnosed with ASD. Four control rhesus monkeyswere exposed to human IgG collected from mothers of multiple typically developing children. Five additional monkeys wereuntreated controls. Monkeys were observed in a variety of behavioral paradigms involving unique social situations. Behaviors werescored by trained observers and overall activity was monitored with actimeters. Rhesus monkeys gestationally exposed to IgG classantibodies from mothers of children with ASD consistently demonstrated increased whole-body stereotypies across multiple testingparadigms. These monkeys were also hyperactive compared to controls. Treatment with IgG purified from mothers of typicallydeveloping children did not induce stereotypical or hyperactive behaviors. These findings support the potential for an autoimmuneetiology in a subgroup of patients with neurodevelopmental disorders. This research raises the prospect of prenatal evaluation forneurodevelopmental risk factors and the potential for preventative therapeutics.? 2008 Elsevier Inc. All rights reserved.Keywords: Repetitive; Primate; Macaque; Macaca mulatta; Activity; Asperger syndrome1. IntroductionAbnormalities in both adaptive and innate immuneresponses have been reported in subjects with autism fornearly 30 years. These include suppressed cell-mediatedand humoral responses to pathogens (Stubbs and Craw-ford, 1977; Warren et al., 1987; Warren et al., 1986), andactive inflammation and autoantibody reactions to brain0889-1591/$ - see front matter ? 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.bbi.2007.12.007qPlease see Brief Commentary by Robert Dantzer and Keith W. Kelleyin this issue.*Corresponding author. Address: The M.I.N.D. Institute, University ofCalifornia-Davis, 2825 50th Street, Sacramento, CA 95817, USA. Fax: +1916 703 0287.E-mail address: dgamaral@ucdavis.edu (D.G. Amaral).1Present address: Department of Psychology, Azusa Pacific University,Azusa, CA 91702-7000, USAwww.elsevier.com/locate/ybrbiAvailable online at www.sciencedirect.comBrain, Behavior, and Immunity 22 (2008) 806–816BRAIN,BEHAVIOR,and IMMUNITY

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tissue or proteins (Ashwood and Van de Water, 2004; Var-gas et al., 2005; Weizman et al., 1982). Neural proteinsreported to be the target of autoantibodies in children withautism include the serotonin receptor (Todd and Ciara-nello, 1985), myelin basic protein (Singh et al., 1993), neu-ron axon filament protein (Singh et al., 1997), cerebellarneurofilaments (Plioplys et al., 1994), and a-2-adrenergicbinding sites (Cook et al., 1993).In addition to the presence of autoantibodies in chil-dren with ASD, antibodies from the serum of somemothers of ASD children have been shown to react toantigens on lymphocytes from their affected children(Warren et al., 1990). Moreover, Dalton et al. (2003)demonstrated the presence of antibodies against brainprotein in the serum of a mother of a child with autism.The exposure of pregnant mice to this mother’s serumresulted in offspring with behavioral abnormalities. Amore exhaustive evaluation of maternally derived serumhas recently been published by Zimmerman et al.(2007). They found a unique pattern of serum reactivityto rat brain from gestational day 18 but not to postnatalday 8 in mothers of children with autism. Similarly, wehave identified a common, characteristic pattern of auto-antibody production to fetal brain protein in the serumof mothers who have had two or more children withASD (Braunschweig et al., 2007). These findings raisethe possibility that a subset of ASD cases are causedby an IgG antibody response directed against the fetalbrain during gestation. To explore this maternal anti-body hypothesis, we exposed pregnant rhesus monkeysto antibodies collected from mothers of children withASD. We compared the behavior of the offspring fromthis group of monkeys to a control group of monkeysprenatally exposed to human IgG from mothers of multi-ple typically developing children and monkeys that wereleft untreated.We chose to use the rhesus monkey as a model for thestudy of autism because it has several specific advantagesover other animal model systems. In particular, the socialrepertoire of monkeys is much broader than that ofrodents making it useful for the analysis of normal andpathological human social behavior (Deaner and Platt,2003; Gothard et al., 2004). Over the past several years,we have developed an extensive battery of behavioraltests aimed at exploring the neural basis of social behav-ior in rhesus monkeys. This battery involves testing dur-ing highlycontrolled socialcomprehensive and well-defined ethogram (catalogue ofspecies-typical behaviors) to quantitatively assess behav-ior. This testing battery has proven to be sensitive atdetecting subtle alterations in social and emotionalbehavior including increased affiliative behavior in adultmonkeys with bilateral lesions of the amygdala andincreased fear responses in infant monkeys with bilaterallesions of the amygdala during novel dyadic social inter-actions (Bauman et al., 2004a, 2004b; Emery et al.,2001).interactions usinga2. Methods2.1. Antibody acquisitionHuman sera from 21 mothers of at least one child with autism and oneor more additional children with autism spectrum disorders were pur-chased from the Autism Genetic Resource Exchange (Mothers of autisticchildren—MAC IgG). Human sera from 7 mothers of multiple typicallydeveloping children were collected locally (Mothers of typically developingchildren—MTDC IgG).2.2. Western blotsAll serum samples were screened for the presence of antibodies directedagainstfetalbraintissueusingWesternblots.Humanfetalbrainproteinmed-ley (300 lg/gel; Clontech Laboratories, Mountain View, CA) or neonatalmonkey brain protein (acquired from the California National PrimateResearchCenter) waspreparedaccordingtotheprotocolusedforthe humanfetal brain as provided by Clontech Laboratories. The monkey brain proteinextract was similarly used at 300 lg/gel. Protein samples were separated on a4–15% gradient reducing gel using SDS–PAGE electrophoresis and trans-ferredontonitrocellulosepaper.Antibodyreactivitytothefetalbrainextractswas then analyzed for all samples individually by Western blotting. The blotswereincubatedfor3 hina0.1 Mphosphatebufferedsaline(PBS,pH7.4)solu-tioncontainingserum from the above samples at a dilution of 1:400, with5%milk and 0.3% Triton X-100. Following a series of rinses, the extracts wereincubated for 1 h with a goat anti-human IgG peroxidase-conjugated anti-body and visualized using chemiluminescence with a Fluorchem 8900 imager(Alpha Innotech, San Leandro, CA). IgG was purified and pooled from 12maternalsamplesderivedfrommothersofautisticchildrenthatdemonstratedone or more bands to human fetal brain proteins which were absent in serumfromcontrolmothers. IgG fromthe 7 control samplesdid not show the reac-tivity to the brain extracts and were also pooled.2.3. Purification of IgG antibodiesEachcollectionofpooledserawasdilutedwithImmunopure(G)IgGbind-ing buffer (Pierce Biotechnology, Inc., Rockford, IL) and IgG antibodies werepurifiedonUltralinkAffinityPackimmobilizedproteinGcolumns(PierceBio-technology,Inc,Rockford,IL).PurifiedIgGwasthenelutedfromcolumnswithImmunopure IgG elution buffer (Pierce Biotechnology, Inc., Rockford, IL).This process resulted in approximately 3.3 mg of purified IgG per 1 ml serum.The purified serum was screened for the presence of HIV and Hepatitis B andC and finally sterile filtered with a 0.2 lm filter prior to injection.2.4. Subjects and living conditionsAll procedures carried out on animal subjects were approved by theUC Davis Institutional Animal Care and Use Committee. The subjectsfor this study were 13 naturally born rhesus monkeys (Macaca mulatta).The mothers of the subjects were selected based on their proven birthrecord and on their high quality maternal behavior and randomly assignedto one of three conditions.Pregnantrhesusmonkeys(n = 4)wereexposedtopurifiedIgG(theonlyantibodies that cross the placental barrier) pooled from the serum of a sub-setofmothersofchildrenwithASDthatcouldbedistinguishedbythepres-enceofreactivityto fetal brainproteinsbyWesternblot(Fig. 1). A separategroup of pregnant monkeys (n = 4) were exposed to purified IgG pooledfrom the serum of mothers of typically developing children. In all cases,15–20 mgofpurifiedIgGdilutedin5 mlofsterilesalinewasdeliveredintra-venouslyon three separate occasions:days 27, 41, and 55 of gestation. Rhe-sus monkey gestation is approximately 165 days. Additional pregnantrhesus monkeys (n = 5) comprised an untreated control group.All infants were born and raised in standard home cages (61 ? 66 ?81 cm). Each mother–infant pair was assigned to one of three socializationcohorts consisting of 6 mother–infant pairs and 1 adult male. There were 2L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816807

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maleand4femaleinfantsineachcohort.Mother–infantpairsfromeachstudygroup were distributed across the socialization cohorts so that there was atleast 1 MAC IgG treated monkey, 1 MTDC IgG control monkey and 1untreatedcontrolmonkeyineachcohort.Inadditiontothe13monkeysinthisstudy,thesocializationcohortsincluded5othermother–infantpairsthatwerenot part of this study. Offspringwere thus raisedwith their mothers andweresocialized for 3 h daily with 5 other mother–infant pairs and 1 adult male inlarge group cages (2.13 ? 3.35 ? 2.44 m). Formal assessments of dominancewithin each socialization cohort indicated that the average dominance rank-ings of the mothers from each study group were roughly equivalent (MACIgG treated = 4.25/6, MTDC IgG control = 3/6, Untreated control = 4/6).When the youngest subject within each socialization cohort reached ?6monthsofage,alloftheinfantswithinthatcohortwerepermanentlyseparatedfrom their mothers (weaned), a standard practice at the primate center, andpermanentlymovedtolargegroupcages.Theadultmalesremainedwitheachcohort and a novel adult female was added to each cohort for a period of 1month following weaning to promote group stability.As anticipated, behavioral data from the control IgG monkeys and theuntreated control monkeys were very similar and did not approach signif-icance. These two groups were therefore pooled to form a single controlgroup (n = 9) for comparisons with the MAC IgG treated monkeys.2.5. Behavioral observationsBehavioral data were collected with The Observer software (Noldus,Sterling, VA; (Noldus, 1991) by three trained observers, demonstratingan inter-observer reliability of at least 85% (agreements/[agreements + dis-agreements] ? 100). All observers had previous experience using Noldussoftware for the collection of rhesus macaque behavioral data (9 months,2 years and 3 years, respectively). Reliability was evaluated using the mostexperienced behavioral observer as the reliability standard. Inter-rater reli-ability was attained for each behavioral task, with observers meeting atleast 85% reliability over two consecutive days. All observers were blindto the experimental status of all subjects.2.6. Preweaning social group and dyad observationsBeginning at approximately one month of age, the infants wereobserved in their socialization cohorts. Each subject was observed for5 min twice per week during weeks in which no other testing took place.One month prior to weaning, two mother–infant pairs were placedtogether in the large testing enclosures for a 20 min tetradic social interac-tion (Bauman et al., 2004a). Each mother–infant pair was observed inpairings with every other mother–infant pair in its own socializationcohort. For both the social group and mother–infant pairings, trainedobservers blind to the condition of the animals used the Observer softwareprogram on laptop computers to score the behavior of each subject in realtime during 5 min focused observations (ethograms of behaviors recordedin the social groups and the familiar dyads have been provided as supple-mentary material).2.6.1. Mother preference testOn the first 4 days immediately following weaning, each infant wasobserved in a test designed to evaluate one aspect of mother–infant attach-ment, the infant’s preference for its mother over another familiar adultfemale (Bauman et al., 2004a). Five daily 2 min trials were conducted, witheach trial consisting of a choice between the infant’s mother and one of thefive other adult females from the infant’s socialization group (the stimulusfemale). A different stimulus female was used for each trial in a predeter-mined pseudo-random order. Before each trial, the test subject was hand-caught by a technician and placed in a plastic release box in the center ofanunfamiliarchainlinktestingenclosure(5.56 ? 1.91 ? 2.13 m).The frontof the subject’s release box was transparent and the remaining three sideswere opaque allowing the test subject to view only the observers untilreleased. The subject’s mother was placed in one of two holding cages,located at either end of the testing enclosure, and the other female wasplaced in the opposite holding cage (holding cage assignments were bal-anced across trials). For the safety of the infant, transparent plastic panelsprevented physical contact between the test subject and the adult females.At the onset of the trial, the subject’s release box and the opaque panelsin front of the holding cages were raised simultaneously, allowing the testsubject to freely move around the testing enclosure and see both its motherand the other female. During each 2 min trial, trained observers recordedthe behaviors exhibited by the test subject, including which adult was firstapproached(scoredwhenthesubjectmovedwithina1 mhalf-circlepaintedon the floor in front of each holding cage. An ethogram of behaviorsfor thematernal preference testing has been provided as supplementary material).2.6.2. Solo and familiar dyad observationsOne month following weaning (when the animals were on average 8.5months old), each subject was observed in a test setting designed to studythe behavior of the subject alone and during interactions with familiarpeers (Bauman et al 2004b; Emery et al 2001). Subjects were removed fromFig. 1. Western blot demonstrating reactivity of maternal serum against both human (HU) and monkey (MO) fetal brain proteins. Depicted are tworepresentative samples from the mothers of multiple children with autism (AU) demonstrating the typical patterns of reactivity noted in the samples usedfor the IV injection of maternal IgG. Note the reactivity in the first AU sample to a band at approximately 60 kDa (left arrow), while the second AUsample reacts to the 60 kDa band as well as a band at 73 kDa (right arrow). Plasma from a representative mother of two typically developing children(TD) lacks a response to either of these two bands. Note that the patterns of reactivity for the human and monkey brain blots are quite similar.808L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816

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their socialization cohorts and placed alone in a large testing enclosure sim-ilar to their home environment and observed for two consecutive 5 min ses-sions. Immediately following these initial solo observations, the first day offamiliar social dyad observations began. Subjects were placed into the test-ing enclosures in pairs to form social dyads. Each social dyad consisted oftwo subjects from the same socialization cohort. Social dyad sessions were20 min in duration with the focal subject (the subject for whom data werecollected) alternating every 5 min. Each subject met with each other subjectin their socialization cohort on two occasions separated by at least one day.Social dyads were spread out over 5 consecutive testing days with eachsubject participating in 2 separate 20 min dyads each day in a predeter-mined pseudo-random order. On the final day of familiar social dyad test-ing, each subject was again observed alone for two consecutive 5 minsessions. Trained observers blind to the condition of the animals used theObserver software program on laptop computers to score the behavior ofeach focal subject in real time from a behavioral ethogram of approxi-mately 50 normal and abnormal rhesus monkey behaviors (ethogram ofbehaviors has been provided as supplementary material). Behaviorsincluded whole-body stereotypies such as pacing, back-flipping, twirling,and swinging. When a subject was engaged in a particular whole-body ste-reotypy for longer than 6 s, an extended stereotypy was scored.2.6.3. Unfamiliar dyad observationsOnemonthfollowingthesolo andfamiliardyad observations (whentheanimalswereonaverage9.5monthsold),eachsubjectwasobservedinatestsetting designed to study interactions with unfamiliar peers. Four age-appropriate monkeys (2 males and 2 females) served as unfamiliar peers(stimulus monkeys). These stimulus monkeys were maternally reared andsocialized in group housing prior to their temporary assignment to thisstudy. Subjects were again removed from their socialization cohorts andplaced in individual holding cages. Subjects were then paired with one ofthe four stimulus monkeys in the same testing enclosures used for soloand familiar dyad observations. These unfamiliar dyad sessions were also20 min in duration with the focal subject alternating every 5 min. Each sub-jectmetwitheachofthestimulusmonkeysontwooccasionsseparatedbyatleast one day. The unfamiliar social dyads were therefore spread out over 4consecutive testing days, with each subject participating in 2 separate20 min dyads each day, again in a predetermined pseudo-random order.2.6.4. Social group observationsIn addition to the acquisition of behavioral data in novel testing envi-ronments, each subject was also observed within their home cage sociali-zation cohorts. Each subject was observed for 5 min twice per weekduring weeks in which no other testing took place. A total of 30 observa-tions were conducted on each subject, with all testing taking place in theweeks following the unfamiliar dyads.2.6.5. Activity monitoringWhen the monkeys were approximately 1 year old, activity was mea-sured with an actimeter (Actiwatch-64; MiniMitter, Bend, OR) housedin a metal casing (40 ? 32 ? 13 mm) attached to a nylon primate collar(Primate Products, Woodside, CA). The Actiwatch-64 is a small device(17 g) capable of detecting the degree and speed of omnidirectional motionwith an accelerometer. Changes in the degree and speed of motion pro-duce changes in voltage that are stored as activity counts. For this study,each actimeter was programmed to sample activity at a frequency of 32 Hzand record activity counts in 30 s intervals. Each monkey was fitted withthe primate collars with the attached actimeters and allowed to acclimateto the collars for 1 week before monitoring began.The activity of each monkey was monitored in two separate condi-tions. In the first, the activity of each monkey was monitored for 7 consec-utive days while the monkeys remained in their routine social housingsituation. In the second condition, the monkeys were removed from theirsocial groups and their activity was monitored while they were individuallyhoused in standard home cages (61 ? 66 ? 81 cm) over two separate 24 hperiods. During each 24 h period, each monkey was observed over fourseparate 10 min sessions, two between 8 a.m. and 12 p.m. and two between3 p.m. and 6 p.m. Behaviors were scored in real time using the same eth-ogram as the solo and dyadic observations. In between each individualhousing period, the monkeys were returned to their social groups for24 h. During both monitoring conditions, special precautions were madeto minimize disturbances to the monkeys.2.7. Statistical analysesDue to the large number of zero values scored for some of the behav-ioral observations, the data were not normally distributed and the vari-ance was not homogenous. We therefore used nonparametric Mann–Whitney tests with a .05 alpha level to determine between-groups differ-ences in these data. For all data in which parametric test assumptions ofnormality and homogeneity of variance were not violated, Independentt-tests with a .05 alpha level were used. All statistical procedures were car-ried out using SPSS 14.0 statistical software.3. Results3.1. Social behaviorsFormal assessments of social behavior during prewea-ning and postweaning observation periods yielded veryfew differences between treated and control animals. Infact, the frequencies of social and nonsocial events weresimilar in the MAC IgG and control groups. When activi-ties characterized as states (extending beyond 3 s) wereanalyzed, there were again only a few differences in socialstates. As summarized in Table 1, important features ofthe maternal infant interaction, such as the amount of ven-tral–ventral contact was also similar in both groups. None-theless, the MAC IgG treated monkeys did demonstrate afew differences in social behavior states during this period.For example, compared to controls, they demonstrated sig-nificantly more contact with their mother and peers priorto weaning (with the exception of ventral contact). How-ever, they demonstrated less contact with familiar peersin the months following the removal of their mothers. Dur-ing this period they also spent significantly more timeengaged in nonsocial active behavior than controls. It ispossible that the MAC IgG treatment was causally relatedto these changes in social behavior and we will explore thisrelationship during future replication studies with largercohorts of treated animals. The most profound observa-tions during the course of this study, however, were ofthe high level of clearly abnormal stereotypic behaviorsexhibited by the MAC IgG monkeys. The remainder of thisreport highlights these differences in stereotypic behaviorsobserved across multiple behavioral settings as well as theincreased activity demonstrated by these same monkeys.3.2. Appearance of stereotypies during the mother preferencetaskResults from the mother preference task showed no sig-nificant differences between the infant monkeys in their ini-tial approach towards their mother or a familiar adultfemale (Mother: t(11) = .423, p = ns; Familiar AdultFemale: t(11) = .107, p = ns). As with control monkeys,the MAC IgG treated monkeys tended to approach theirL.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816809

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own mother first. However, a unique pattern of pacingacross the length of the testing cage was observed in infantsexposed to IgG from mothers of children with autism (SeeSupplementary Video 1). The Mann–Whitney test revealed that the number of pacing episodes was significantly higherfor these monkeys compared to controls (U = 6.00,p = .024, Fig. 2). This behavior stood out to our trainedobservers as being highly unusual for socially reared mon- keys and prompted a more intensive analysis of motoractivityandstereotypedbehaviorsinothertestingconditions. 3.3. Presence of stereotypies during solo observations andfamiliar pairingsOne month following the mother preference task, eachsubject was removed from their social group and observed either alone or with a familiar partner in a large novelenclosure that was similar to their home environment.Nonparametric statistics demonstrated significant differ-ences in the frequency of whole-body stereotypies betweengroups when the animals were alone (U = 5.00, p = .014).Fig. 3 shows that the MAC IgG treated monkeys showed elevated frequencies of whole-body stereotypies comparedto the control monkeys. A similar pattern of elevated ste-reotypies was also apparent when the monkeys were paired with a familiar partner (Fig. 4). In this case, the frequencyof stereotypies demonstrated a tendency to be higher in theMAC IgG treated animals (U = 7.00, p = .076). However,both the frequency and duration of bouts of extended ste- reotypy were clearly significantly higher in this paired con-dition (Frequency: U = 5.00, p = .014; Duration: U = 5.00,p = .014). Supplementary Videos 2 and 3 demonstrate theTable 1Mean duration (s) of social states for MAC IgG and control monkeys across social testing paradigmsBehavioral StatePrewean Mother–infant dyads Prewean social groups Postwean familiar dyadsPostwean unfamiliar dyads Postwean social groupsMAC IgGControlMAC IgG ControlMAC IgG Control MAC IgG Control MAC IgGControlMean SEM MeanSEMMeanSEMMean SEMMean SEM MeanSEM Mean SEMMeanSEM Mean SEMMeanSEMBreast contactVentral contactOther contactExtended playNonsocial activeProximityExtended groomExtended toy playExtended contactSleep619.53 217.131109.88 318.96 1293.52 147.081482.92 148.02 1170.40 106.93 1585.67 124.37 1162.91 102.1826.2511.59 103.8929.061451.23 402.39 1932.41 232.81 1687.75 476.09 2592.41 284.81 4235.61 186.10 3430.68 239.00 4055.24 139.54 4088.95 116.86 4357.641272.67 176.71 1142.0567.08 1276.85 381.07 1495.93 115.51832.30 180.92 1078.92****0.000.004.53 2.62 36.93** ** ****15.706.41 11.913.517.12********353.19 125.09********22.28312.04 73.02682.95 258.57327.65 286.77446.94 183.37461.24 194.48************************************167.2682.73 365.3994.593.733.7311.003.7544.4232.6533.5414.64 161.8730.6290.52 3658.96 195.9883.69 983.7849.31294.2849.23158.89339.0174.7182.4226.0024.11313.656.443.76161.29**68.614.843.7639.05**369.0867.6619.84135.42**67.4736.547.5735.98 1018.86 105.89 1071.86 122.10**470.60 163.41821.30173.39105.6955.4985.0639.3736.930.3646.3077.62811.80 113.1423.34 20.619.91 551.4286.97There were no differences in important maternal–infant interactions such as ventral–ventral contact. During the preweaning social groups, MAC IgG treated infants spent significantly more time incontact with their mother and peers than control infants (t(11) = 2.410, p = .035). During the postweaning familiar dyads, MAC IgG treated monkeys spent significantly less time in the extendedcontact state with their peers (t(11) = 2.403, p = .035) and showed a trend towards more time in a nonsocial active state than control monkeys (t(11) = 2.088, p = .061). The increase in nonsocial activebehavior was also observed in postweaning social groups (t(11) = 2.279, p = .044).*Indicates that the social state was not applicable to the testing paradigm.**Indicates insufficient data due to the rareoccurrence of the social state within the testing paradigm. Bold font indicates significant differences between control and MAC IgG monkeys.Fig. 2. Mean episodes of pacing behavior observed during the mother preference task in control monkeys (both untreated controls and animals treated with IgG from mothers of typically developing children) comparedto MAC IgG treated monkeys. The MAC IgG treated monkeysdemonstratedsignificantlymorepacingepisodesthancontrols(U = 6.00, p = .024). Error bars represent SEM. Diamonds here, and inFigs. 3–5 and 7–8, represent scores of individual animals in each group. Insome cases, scores are so similar that symbols overlap.810L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816

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pacing and back-flipping behavior observed during thepairing with a familiar partner.3.4. Presence of stereotypies during unfamiliar pairingsMonkeys were also observed for behavioral differenceswhen paired with unfamiliar peers in dyadic situations.One month following the pairings with familiar peers,monkeys were removed from their social groups and placedwith one of four unfamiliar ‘‘stimulus” monkeys in thesame large testing enclosures. Once again, we observed asignificant difference in the frequency of whole-body ste-reotypies between the groups (U = 5.50, p = .028). The ele-vated stereotypies of the MAC IgG treated monkeyscompared to control monkeys are illustrated in Fig. 5.3.5. Elevated levels of activity during solo housingIndependent t-tests did not reveal any significant differ-ences in activity between control and MAC IgG monkeysover the 7 days of monitoring during routine social housingFig. 3. Mean episodes of whole-body stereotypies observed in control(both untreated controls and animals treated with IgG from mothers oftypically developing children) and MAC IgG treated monkeys during soloobservations. The MAC IgG treated monkeys displayed significantly moreepisodes of whole-body stereotypies than controls (U = 5.00, p = .014).Error bars represent SEM.Fig. 4. Mean episodes of whole-body stereotypies observed in control(both untreated controls and animals treated with IgG from mothers oftypically developing children) and MAC IgG treated monkeys duringobservations with a familiar partner. The MAC IgG treated monkeysdisplayed more episodes of whole-body stereotypies than control mon-keys, although the results only approached significance (U = 7.00,p = .076). Data analysis of the number and duration of extendedstereotypy bouts were significant during this task (frequency: U = 5.00,p = .014; Duration: U = 5.00, p = .014). Error bars represent SEM.Fig. 5. Mean episodes of whole-body stereotypies observed in control(both untreated controls and animals treated with IgG from mothers oftypically developing children) and MAC IgG treated monkeys duringobservations with an unfamiliar partner. The MAC IgG treated monkeysdisplayed significantly more episodes of whole-body stereotypies thancontrol monkeys (U = 5.50, p = .028). Error bars represent SEM.Fig. 6. Mean number of activity counts (arbitrary units) per 30 s ofmonitoring for control (both untreated controls and animals treated withIgG from mothers of typically developing children) and MAC IgGmonkeys during day and night combined, day only, and night only, inboth social housing and individual housing conditions. There were nodifferences in activity demonstrated during the social housing condition.When the monkeys were removed from their social cages and placed intoindividual housing, significantly higher activity counts were recorded forthe MAC IgG treated monkeys (day and night combined: t(11) = 2.954,p = .013). Error bars represent SEM.L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816811

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(Fig. 6). However, during the individual housing condition,MAC IgG monkeys demonstrated significantly more activ-ity than controls (t(11) = 2.954, p = .013; Fig. 6). Furtheranalysis showed that this increase in activity was primarilycaused by higher activity levels during the day (light cycle;t(11) = 2.758, p = .019) rather than at night (dark cycle;t(11) = 1.951, p = .077).Behavioral observations during solo housing againrevealed significantly more whole-body stereotypies inMAC IgG monkeys compared to controls (U = 0.00,p = .003; Fig. 7). As it is reasonable to assume that the ele-vated activity of the MAC IgG monkeys is related to theirwhole-body stereotypies, we compared their activity countsto their whole-body stereotypies recorded during eight10 min observations. The monkey with the highest numberof activity counts actually had the second lowest number ofstereotypies (Fig. 8). In addition, there was less than 5%difference in the activity counts of all of the MAC IgGmonkeys, but there was a 187% increase in the number ofstereotypies from the lowest count to the highest. There-fore, there did not appear to be any causal relationshipbetween the whole-body stereotypies and elevated activity.4. DiscussionTo summarize, the group of monkeys exposed prena-tally to IgG from mothers of children with ASD demon-strated significantly more stereotypies and higher levels ofmotor activity than control monkeys. The elevation in ste-reotypies was first evident during the mother preferencetask that was conducted in the first 4 days following thepermanent removal of the subjects’ mothers from thesocialization groups. The increase in episodes of stereo-typed behavior persisted and strengthened in the sixmonths following weaning. Importantly, significantlyincreased stereotypies were observed consistently acrossfive different testing paradigms: mother preference testing,solo observations, familiar dyadic interactions, unfamiliardyadic interactions, and during solo activity monitoring.In addition to quantifying stereotyped behaviors, wealso used actimiters to monitor the general activity of theanimals at approximately one year of age. Significant eleva-tion of activity was evident when the monkeys were indi-viduallyhoused,but notenvironment. It is important to point out that the rise inactivity did not appear to be caused by the presence of ste-reotypies. A scatterplot of stereotypy and activity data col-lected over eight 10 min blocks indicated that thecontribution of the stereotypies to the overall measure ofactivity was minimal (Fig. 8) and thus the presence of ste-reotypies could not account for the 1.47 fold increase inactivity observed between the MAC IgG treated and con-trol monkeys (Fig. 6).intheir normalsocial4.1. Exposure to MAC IgG appears to cause thestereotypical behavior observed in the experimental monkeysStereotypical behavior in monkeys is a sign of pathol-ogy. It can be brought on by, among other things, long-term individual housing conditions or environmentalimpoverishment (Lutz et al., 2003; Mason, 1991). Neitherof these conditions applies to our experimental subjects.The monkeys in this study were maternally reared andsocialized with other mother–infant pairs on a daily basisprior to weaning, and permanently socialized with 5 othermonkeys following weaning. It is important to point outthat the much more invasive procedure of producing neo-Fig. 7. Mean episodes of whole-body stereotypies observed in control(both untreated controls and animals treated with IgG from mothers oftypically developing children) and MAC IgG treated monkeys over eight10 min observation periods occurring during solo activity monitoring. Thedata are shown per 5 min of observation for comparison with previousfigures. The MAC IgG treated monkeys displayed significantly moreepisodes of whole-body stereotypies than control monkeys (U = 0.00,p = .003). Error bars represent SEM.Fig. 8. Scatterplot of the whole-body stereotypy and activity count dataof the 4 MAC IgG treated monkeys collected during eight 10 minobservation periods. The numbers of stereotypies do not appear to beclosely related to the activity counts indicating that the stereotypicmovements were largely independent of generalized activity.812L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816

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natal lesions of the amygdala or hippocampal formation(Bauman et al 2004a; Bauman et al 2004b) did not resultin the production of increased whole body stereotypies dur-ing the first year of life. As an added precaution in the cur-rent study, each social cohort included representation fromeach treatment group so it is unlikely that the increased ste-reotypies were learned through social transmission. There-fore, given that these stereotypies were only observed in theMAC IgG treatment group, they appear to be attributableto that particular form of IgG exposure. It is also impor-tant to note that no differences were observed betweenuntreated control animals and animals treated with IgGfrom mothers of typically developing children.4.2. Potential exacerbation of stereotypies and hyperactivityby novel social and environmental conditionsThestereotypiesobservedintheMACIgGmonkeyswerenot as apparent prior to weaning, and, while occasionallyobserved in their home cages, were not consistently presentin their routine living condition. Rather, the stereotypiesemergedwhenthemonkeyswereremovedfromtheirnormalenvironment andplaced in anovelsocial setting. It isimpor-tanttoemphasizethatthecontrolmonkeyshadpreciselythesame rearing conditions as the MAC IgG group.Stereotypieshavebeenconsideredoneofthedefiningfea-tures of autism since the earliest descriptive account (Kan-ner, 1943). Along with the presence of stereotypies,individuals with autism are often described as having an‘‘insistenceonsameness”intheirenvironment.Afewstudieshave explored the effects of the environment on the rates ofstereotyped behavior in autism. One study reported a pro-gressive increase in stereotypies as unfamiliar toys followedby an unfamiliar passive adult were introduced into anempty room (Hutt, 1965). Another study demonstrated asignificant increase in stereotypies with an unfamiliar versusa familiar therapist (Runco et al., 1986). Given these obser-vations of children with autism, it is noteworthy that thetreated monkeys in our study did not demonstrate increasedstereotypical behavior in their long-term, stable socialgroups,but only innovelenvironmental orsocial situations.Interestingly, the significantly elevated levels of activityfound in the treated monkeys were also only present afterthey were removed from their normal social environmentand housed individually in a novel environment. Althoughhyperactivity is not a defining feature of autism, it is one ofthe most frequently reported problems in ASD (Lecavalier,2006). In fact, recent studies have indicated that approxi-mately 50% of children with ASD meet DSM-IV symptomcriteria for ADHD (Gadow and DeVincent, 2005; Gadowet al., 2004). It is likely that changes in social environmentsexacerbate these symptoms.4.3. Caveats of the current modelWhile the neurodevelopmental alterations that havebeen produced in this population of animals are striking,it is premature to conclude that this is an animal modelof autism. We have thus far not found any profound differ-ences in social behavior between groups as one might pre-dict in a monkey model of autism. We believe that this maybe due to experimental limitations of the model. First, dueto limited availability of IgG, we were only able to exposethe pregnant females to IgG for approximately 25% of theduration of the pregnancy. Second, the time during preg-nancy when the females were exposed may determine thetype of behavioral pathology that arises. Our maternalantibody hypothesis of human autism is based on the pre-mise that before or during pregnancy, the maternalimmune system recognizes one or more unidentified fetalbrain proteins as foreign and mounts an antibody responseagainst it. This, therefore, involves an active immune pro-cess with a constant source of the pathologic antibody.In other words, it is likely that the donor mothers had titreof these autoantibodies during most of their pregnancy.Interestingly, of the 12 mothers who contributed serumto the MAC IgG group, there were no typical children bornafter their first child with ASD, potentially indicative of apersistent pathological immune response. We chose to deli-ver the IgG in three different doses spanning the end of thefirst trimester of pregnancy based upon the demonstratedsensitivity of the nervous system during this period ofdevelopment in the monkey (Hendrickx, 1973; Hendrickxet al., 1988), and during the equivalent time of neural devel-opment in rodent models (Rodier et al., 1997; Shi et al.,2003). However, we were limited from producing a longerexposure by the quantity of available IgG and the half-lifeof human IgG in the rhesus monkey (Hinton et al., 2004).We would predict that a longer exposure of the fetus to theIgG may either increase the severity of the stereotypicaland hyperactive behaviors or produce impairments ofsocial behavior that are characteristic of autism. This willbe addressed in future studies.4.4. Precedents for neonatal disorders caused throughplacental transfer of IgGThere are a number of examples in the literature ofpediatric disorders caused by transplacental transfer ofmaternally derived IgG. Transplacental transfer of anti-acetylcholine receptor antibody, for example, can producesymptoms of myasthenia gravis in the newborn even if themother is in remission during pregnancy (Elias et al., 1979).Some mothers with myasthenia gravis who carry highlyspecific antibodies to a fetal isoform of the nicotinic acetyl-choline receptor give birth to children with arthrogryposismultiplex congenital (AMC; Brueton et al., 2000; Vincentet al., 1995). AMC is a severe developmental conditioninvolving fixed joint contractures and other deformitiesthat results from lack of movement. In anti-AChR anti-body-associated AMC, fetal or neonatal death is commonand the condition usually recurs in the mother’s subsequentpregnancies (Polizzi et al., 2000). Administration of serumfrom such mothers to pregnant mice results in offspringL.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816813

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with fixed joint contractures and other developmentalabnormalities (Jacobson et al., 1999).Anotherexample, Graves’production of antibodies against the thyroid stimulatinghormone receptor (TSHr) in the thyroid gland. Theantibodies appear to act as a receptor agonist since symp-toms of Graves’ disease are identical to the symptoms ofhyperthyroidism and include an enlarged thyroid, nervous-ness, heat intolerance, weight loss, sweating, tremors, heartpalpitations and exophthalmos. During pregnancy, anti-bodies to TSHr cross the placenta and affect the thyroidgland of the fetus (Radetti et al., 1999; Volpe et al.,1984). Infants born to mothers with Graves’ disease oftenshow signs similar to those of the mother; the symptomsdisappear as the maternal antibodies are eliminated. Thereare a number of other transient neonatal autoimmunediseases (reviewed in Giacoia and Azubuike, 1991) whicheffect the central or periphereral nervous system. Thus,there is adequate precedent for the hypothesis that mater-nally derived antibodies to fetal brain protein may crossthe placenta, interact with the developing fetal brain andaffect the outcome of the developmental process.disorder,involves the4.5. Evidence from experimental animal and clinical researchfor immune modulation of cognitive and motor functionSystemic lupus erythematosous (LSE) is an autoim-mune disorder that affects multiple body systems. In anelegant series of studies, Diamond, Volpe and colleagueshave examined the role of serum antibodies specific forDNAandfortheN-methly(NMDAr) in the neuropsychiatric and cognitive symp-toms associated with LSE (Diamond et al., 2006; Kowalet al., 2006). They have injected serum from patients con-taining these antibodies into mature mice who are alsotreated with lipopolysacharide to cause a breach of theblood brain barrier. They find that the brains of treatedanimals show neuronal pathology in the CA1 region ofthe hippocampus and are impaired on hippocampal-dependent memory tasks. They have raised the hypothesisthat lupus neuropsychiatric symptoms are caused by adirect action of the antibodies on brain function. Consis-tent with this, they found that the postmortem brains ofpatients with SLE demonstrate the anti NMDAr and antiDNA antibodies. Interestingly, if the breach of the bloodbrain barrier leads to the antibodies interacting with aspecific brain region such as the amygdala, then theresulting behavioral pathology can be quite different(Emmer et al., 2006).There is also substantial support for the idea thatimmune modulation of the brain can lead to pathologicalmotor behaviors. An excellent example of this is the workof Zalcman et al. (1999), Zalcman (2001). They have found,for example, that a single intraperitoneal injection of IL-2produces significantly increased locomotor and exploratorybehavior in mice in a novel environment. Interestingly, IL-2 treatment also significantly increased sensitivity to theD-aspartate receptorbehavioral stimulating properties of GBR 12909 which isa highly selective dopamine uptake inhibitor. This raisesthe possibility that IL-2 acts to increase locomotor activitythrough a dopaminergic mechanism.Finally, the PANDAS (Pediatric Autoimmune Neuro-psychiatric Disorders Associated with Streptococcal infec-tions) provide another good example of how an immuneresponse can produce antibodies that interact with brainand result in abnormal behaviors, including motor andvocal tics. Through what appears to be a molecular mim-icry mechanism, strep infections lead to the production ofautoantibodies that particularly affect the basal ganglia(Snider and Swedo, 2004). Interestingly, antibodies associ-ated with PANDAS have been demonstrated to react withneurons from the caudate-putamen and induce calcium–calmodulin dependent protein kinase II (CaM kinase II)activity. Depletion of serum IgG reduced CaM kinase IIcell signaling and reactivity was blocked by streptococcalantigen N-acetyl-b-D-glucosamine (Kirvan et al., 2006b).A very similar mechanism has been proposed for the devel-opment of Syndeham’s chorea (Kirvan et al., 2006a).While this has not been an exhaustive review, there issubstantial evidence to conclude that an autoimmunemechanism can play a role in the development of pediatricdisorders and disorders that manifest with cognitive andbehavioral alterations. Given the demonstration of unusualfetal brain-directed autoantibodies in the serum of somewomen who give birth to children with autism (Braun-schweig et al., 2007; Zimmerman et al., 2007) and the cur-rent data of abnormal motor behaviors in rhesus monkeystreated during gestation with IgG from mothers of childrenwith autism, the hypothesis of an autoimmune etiology forat least some cases of autism appears reasonable.4.6. Clinical implications of this researchMindful of the caveats described above, the presentstudy nonetheless raises several important clinical implica-tions. The evaluation of the occurrence of brain-directedautoantibodies could be a useful indication of risk factorsfor autism or other neurodevelopmental disorders inwomen who intend to become pregnant. This would beparticularly relevant to mothers who have already had achild with autism since the risk of a second child with aut-ism increases substantially over that of the general popula-tion.Moreover, bydeterminingantibodies are present, appropriate therapeutic interven-tions could be implemented to lessen the risk of producinga child with a neurodevelopmental disorder. This raises theoptimistic prospect that some future cases of autism orrelated neurodevelopmental disorders may be preventedthrough prenatal diagnostic screening.that brain-directedAcknowledgmentsWe thank Jennifer Forcier, Jessica Toscano, MelissaMarcucci, and Hannah Cuthbert for technical support.814L.A. Martin et al./Brain, Behavior, and Immunity 22 (2008) 806–816